This work was sponsored by the Medicine and Health Technology Plan of Zhejiang Province (2019KY050), the Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province (2019ZA026), the Key Research and Development Plan in Zhejiang Province (Grant No.2020C03043), the Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province (2021ZQ021), and the Zhejiang Provincial Natural Science Foundation of China (LQ22H060007).

This study was approved by the Ethics Committee of Tongde Hospital of Zhejiang Province.
1. Preparation of the DC-ECM hydrogel
NOTE: In this study, the cartilage was obtained from the knee joints of 12 month old Bama miniature pigs, avoiding the collection of bone tissue.
<ol>
	<li>Take the collected cartilage, and block and chop it into 1-2 mm3 pieces with a scalpel.</li>
	<li>Place 20 g of the minced cartilage in a 50 mL centrifuge tu...

<ol>
	<li>Vega, S. L., Kwon, M. Y., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+hydrogels+for+cartilage+tissue+engineering.">Recent advances in hydrogels for cartilage tissue engineering.</a> European Cells &amp; Materials. 33, 59-75 (2017).</li><li>Yang, J., Zhang, Y. S., Yue, K., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-laden+hydrogels+for+osteochondral+and+cartilage+tissue+engineering.">Cell-laden hydrogels for osteochondral and cartilage tissue engineering.</a> Acta Biomaterialia. 57, 1-25 (2017).</li><li>Bejleri, D., Davis, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix+materials+for+cardiac+repair+and+regeneration.">Decellularized extracellular matrix materials for cardiac repair and regeneration.</a> Advanced Healthcare Materials. 8 (5), e1801217 (2019).</li><li>Brown, M., Li, J., Moraes, C., Tabrizian, M., Li-Jessen, N. Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix:+New+promising+and+challenging+biomaterials+for+regenerative+medicine.">Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine.</a> Biomaterials. 289, 121786 (2022).</li><li>Barbulescu, G. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix+scaffolds+for+cardiovascular+tissue+engineering:+Current+techniques+and+challenges.">Decellularized extracellular matrix scaffolds for cardiovascular tissue engineering: Current techniques and challenges.</a> International Journal of Molecular Sciences. 23 (21), 13040 (2022).</li><li>Zhang, W., Du, A., Liu, S., Lv, M., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+in+decellularized+extracellular+matrix-derived+hydrogels.">Research progress in decellularized extracellular matrix-derived hydrogels.</a> Regenerative Therapy. 18, 88-96 (2021).</li><li>Zhu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+decellularized+extracellular+matrix+scaffolds+for+articular+cartilage+repair.">Cell-derived decellularized extracellular matrix scaffolds for articular cartilage repair.</a> International Journal of Artificial Organs. 44 (4), 269-281 (2021).</li><li>Li, T., Javed, R., Ao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenogeneic+decellularized+extracellular+matrix-based+biomaterials+for+peripheral+nerve+repair+and+regeneration.">Xenogeneic decellularized extracellular matrix-based biomaterials for peripheral nerve repair and regeneration.</a> Current Neuropharmacology. 19 (12), 2152-2163 (2021).</li><li>Xia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+cartilage+as+a+prospective+scaffold+for+cartilage+repair.">Decellularized cartilage as a prospective scaffold for cartilage repair.</a> Materials Science &amp; Engineering C-Materials for Biological Applications. 101, 588-595 (2019).</li><li>Chen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desktop-stereolithography+3D+printing+of+a+radially+oriented+extracellular+matrix/mesenchymal+stem+cell+exosome+bioink+for+osteochondral+defect+regeneration.">Desktop-stereolithography 3D printing of a radially oriented extracellular matrix/mesenchymal stem cell exosome bioink for osteochondral defect regeneration.</a> Theranostics. 9 (9), 2439-2459 (2019).</li><li>Saldin, L. T., Cramer, M. C., Velankar, S. S., White, L. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+hydrogels+from+decellularized+tissues:+Structure+and+function.">Extracellular matrix hydrogels from decellularized tissues: Structure and function.</a> Acta Biomaterialia. 49, 1-15 (2017).</li><li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+delivery+in+tissue-specific+hydrogel+enabled+meniscal+repair+in+an+orthotopic+rat+model.">Stem cell delivery in tissue-specific hydrogel enabled meniscal repair in an orthotopic rat model.</a> Biomaterials. 132, 59-71 (2017).</li><li>Zheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intensified+stiffness+and+photodynamic+provocation+in+a+collagen-based+composite+hydrogel+drive+chondrogenesis.">Intensified stiffness and photodynamic provocation in a collagen-based composite hydrogel drive chondrogenesis.</a> Advanced Science. 6 (16), 1900099 (2019).</li><li>Young, J. L., Holle, A. W., Spatz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P.Nanoscale+and+mechanical+properties+of+the+physiological+cell-ECM+microenvironment.">P.Nanoscale and mechanical properties of the physiological cell-ECM microenvironment.</a> Experimental Cell Research. 343 (1), 3-6 (2016).</li><li>Abdolghafoorian, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+heart+valve+decellularization+on+xenograft+rejection.">Effect of heart valve decellularization on xenograft rejection.</a> Experimental and Clinical Transplantation. 15 (3), 329-336 (2017).</li></ol>

The authors have nothing to disclose.

This protocol provides a systematic approach for the preparation of decellularized cartilage extracellular matrix hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical, chemical, and enzymatic disruption to remove cellular material while preserving the structure and composition of the ECM. The protocol's critical steps include adjusting the decellularization time and methods and ensuring complete decellularization.
Compared to other ex...

Cartilage tissue engineering is a rapidly developing field that seeks to regenerate damaged or diseased cartilage tissue1. One key challenge in this field is the development of biomimetic scaffolds that can support the growth and differentiation of chondrocytes, the cells responsible for producing cartilage2. The ECM of cartilage tissue plays a critical role in regulating the behavior of chondrocytes. DC-ECM is an effective scaffold for tissue engineering applications3.
A number of techniques have been developed to produce DC-ECM from cartilage tissue, including chemical, enzymatic, and physical methods. However, these methods often result in the generation of ECM hydrogels that are insufficiently biomimetic, which limits their potential for use in tissue engineering applications4,5. Thus, there is a need for a more effective method for producing DC-ECM hydrogels.
The development of this technique is important because it can advance the field of tissue engineering by providing a new approach for creating biomimetic scaffolds that can support tissue regeneration and repair. Furthermore, this technique could be easily adapted to produce ECM hydrogels from other tissues, thereby expanding its potential applications.
In the broader body of literature, there has been growing interest in using DC-ECM as a scaffold for tissue engineering applications6. Numerous studies have demonstrated the effectiveness of DC-ECM hydrogels in promoting cell growth and differentiation in various tissues, including cartilage7,8. Therefore, the development of a protocol for producing DC-ECM hydrogels that closely mimic the natural ECM of cartilage tissue is a significant contribution to the field.
The protocol presented in this paper addresses this need by providing a novel method for producing DC-ECM hydrogels that closely mimic the natural ECM of cartilage tissue. The protocol involves decellularizing cartilage tissue, isolating the resulting ECM, and creating a hydrogel by cross-linking the ECM with a biocompatible polymer. The resulting hydrogel has shown promising results in supporting the growth and differentiation of chondrocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M Tris-HCl, pH7.6</td>
			<td>Beyotime</td>
			<td>ST776-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH8.0</td>
			<td>Beyotime</td>
			<td>ST780-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C Freezer</td>
			<td>Eppendorf</td>
			<td>F440340034</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease</td>
			<td>Aladdin</td>
			<td>D128600-80KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DNEasy Blood &#38;Tissue Kit</td>
			<td>Qiagen</td>
			<td>No. 69506</td>
			<td></td>
		</tr>
		<tr>
			<td>GAG colorimetric quantitative detection kit</td>
			<td>Shanghai Haling</td>
			<td>HL19236.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HCP-2 dryer&#160;</td>
			<td>Hitachi</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop8000</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Gibco</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonuclease</td>
			<td>Aladdin</td>
			<td>R341325-100 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma500</td>
			<td>ZIESS</td>
			<td>N/A</td>
			<td>Scanning electron microscope</td>
		</tr>
		<tr>
			<td>Spectra S</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Stainless steel sieve</td>
			<td>SHXB-Z-1</td>
			<td>Shanghai Xinbu</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Beyotime</td>
			<td>P0096-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Gibco</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet lamp</td>
			<td>Omnicure 2000</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B2</td>
			<td>Gibco</td>
			<td>R4500-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Shanghai Qiasen</td>
			<td>78HW-1&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of decellularized cartilage extracellular matrix hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

To prepare a better DC-ECM cartilage hydrogel, we studied and reviewed the previous literature and compared the various decellularization protocols in terms of the decellularization ratio, immunogenicity, and mechanical functionality9.
On this basis, we prepared the DC-ECM cartilage hydrogel and explored the effect of a radially oriented extractive matrix/mesenchymal stem cell exosome bio-ink in treating osteochondral defects. The results showed that the DC-ECM cartilag...

Watch this Scientific Journal Video about Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels at JoVE.com

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Our protocol is significant as it enables the creation of disease in hydrogels that closely resemble the nature of cartilage tissue. The main advantage of this technique is ability to produce disease in hydrogels with evident biological activity in spatial structure, low immunogenicity, and biological industrial function. To begin, chop the collected cartilage using a scalpel into pieces of one to two cubic millimeters in size.In a 50-millimeter centrifuge tube, submerge 20 grams of the minced cartilage in 20 milliliters of hypotonic Tris-hydrochloride buffer. Place the tube at 80 degrees Celsius for three hours, and then in the oven at 37 degrees Celsius for three hours. Vortex the centrifuge tube for 30 seconds at 1000 RPM.Then filter the decellularized cartilage using a plastic sieve placed on a 50-milliliter centrifuge tube. Wash the cartilage thrice with sterile PBS before collecting it. Add 10 milliliters of trypsin solution to the collected cartilage and incubate it on a shaker at 37 degrees Celsius for 24 hours, replacing the trypsin every four hours.After filtering the suspension, wash the trypsinized cartilage with hypertonic buffer. Once the cartilage is preserved, add 10 milliliters of nuclease solution and place it on a shaker at 37 degrees Celsius for four hours. After washing with sterile PBS, add hypertonic Tris-hydrochloride solution to the cartilage and place it on the shaker as demonstrated.Once washed with sterile PBS, submerge the tissue in 1%Triton X-100 solution for 24 hours. After removing the Triton X-100, rinse the decellularized cartilage with distilled water for three days, changing the water every 12 hours. After three days, add peracetic acid solution to the cartilage and soak it for four hours.Once washed with PBS, collect the cartilage using a plastic sieve as previously demonstrated. Using liquid nitrogen, prepare decellularized cartilage powder. Add 80 milliliters of 0.5 molar acetic acid and 20 milligrams of pepsin to two grams of cartilage powder, and digest the mixture for 24 hours at 37 degrees Celsius.After digestion, centrifuge the suspension at 400 G for 10 minutes and collect the supernatant, which is now called DC-ECM solution For storing the DC-ECM solution, add one milliliter of the solution to each well of a six-well plate and place it in a lyophilizer. Once the solution is freeze dried, transfer it to a centrifuge tube. To form a hydrogel, dissolve 20 milligrams of freeze dried DC-ECM solution in one milliliter of sterile distilled water.Once vortexed, add one milligram of vitamin B2 to the DC-ECM solution. After incubation, irradiate it with UV light for three minutes. Add buffer GTL and proteinase K to 20 milligrams of DC-ECM cartilage powder, and mix thoroughly using vortex oscillation.For complete cartilage dissolution, incubate the mixture at 56 degrees Celsius for four hours. Once vortexed, add 200 microliters of buffer GTL followed by anhydrous ethanol. After vortexing, centrifuge the sample at 6000 G for one minute at four degrees Celsius.Then collect and quantify the DNA as described in the manuscript. For analyzing collagen, acidify five milligrams of DC-ECM powder in hydrochloric acid at 100 degrees Celsius for 20 minutes. Then neutralize it with five milliliters of six molar sodium hydroxide solution.Calculate the hydroxyproline content of the neutralized sample using absorbance at 570 nanometers against the hydroxyproline standard. Add 500 microliters of reagent A to 200 milligrams of DC-ECM powder after mixing. Incubate the sample at four degrees Celsius for 16 hours.Once centrifuged, collect 50 microliters of sample solution and add 50 microliters of reagent B, followed by reagent C.After incubating the sample for 10 minutes, add 750 microliters of reagent D and incubate for 30 minutes in the dark. After centrifugation, add one microliter of reagent E to the pellet and mix well before centrifuging. Then dissociate the sample before GAG content measurement.In the prepared DC-ECM cartilage, the DNA content was significantly eliminated. However, the collagen and GAG content were retained compared to the native cartilage. The SEM and TEM analysis demonstrated the ultra structure of the prepared DC-ECM.The prepared hydrogel in the inverted tube did not flow to the bottom, thus demonstrating gelation. Further, compared to the DC-ECM only solution, the addition of vitamin B2 reduced the gelation time due to the induced crosslinking during the DC-ECM hydrogel formation. The DC-ECM hydrogel viscosity was higher than the solution viscosity and increased shear rates decreased the solution viscosity.Moreover, the high storage modulus of the DC-ECM solution and hydrogel indicated that they both had gel rather than liquid properties. The SEM analysis demonstrated that the pore size of the DC-ECM solution was significantly decreased in the hydrogel form after crosslinking and freeze drying. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove cellular material while preserving the structure and the conversation of the ECM.

synthesis-of-decellularized-cartilage-extracellular-matrix-hydrogels

Research

JoVE Journal

Bioengineering

779 Görüntüleme.  Tongde Hospital of Zhejiang Province. Protokolümüz, kıkırdak dokusunun doğasına çok benzeyen hidrojellerde hastalık oluşturulmasını sağladığı için önemlidir. Bu tekniğin ana avantajı, mekansal yapıda belirgin biyolojik aktiviteye, düşük immünojenisiteye ve biyolojik endüstriyel fonksiyona sahip hidrojellerde hastalık üretme yeteneğidir. Başlamak için, toplanan kıkırdağı bir neşter kullanarak bir ila iki milimetre küp büyüklüğünde parçalar halinde doğrayın.50 milimetrelik bir sant...